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A switch in heme axial ligation prepares Paracoccus pantotrophus cytochrome cd1 for catalysis

Nature Structural Biology volume 7, pages 885–888 (2000)Cite this article

Abstract

Cytochrome cd1 nitrite reductase (cd1) from Paracoccus pantotrophus is a respiratory enzyme capable of using nitrite, hydroxylamine and oxygen as electron accepting substrates. Structural studies have shown that when the enzyme is reduced there is a change in the axial ligation of both hemes, which has been proposed to form part of the catalytic cycle. Here we report the use of a physiological electron donor, pseudoazurin, to investigate the relationship between heme ligation and catalysis. A combination of visible absorption and electron paramagnetic resonance spectroscopies reveals the formation of a catalytically competent state of oxidized cd1 with ‘switched’ axial ligands immediately after complete reoxidation of reduced cd1 with hydroxylamine. This activated conformer returns over 20 min at 25 °C to the state previously observed for oxidized ‘as isolated’ cd1, which is catalytically inactive towards the same substrates.

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Figure 1: Axial ligand switches that occur at the c-heme and d1-heme centers upon oxidation/reduction of cytochrome cd1 from P. pantotrophus.
Figure 2: The inability of pseudoazurin and ‘as isolated’ P. pantotrophus cytochrome cd1 to exchange electrons can be overcome by chemical pre-reduction of the enzyme.
Figure 3: Spectroscopic probing of structural changes in cytochrome cd1 after reaction with the two-electron oxidant hydroxylamine.
Figure 4: The role of cytochrome cd1 with switched axial heme ligation in the reduction of hydroxylamine.

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Acknowledgements

We would like to thank A. Koppenhöfer for his contribution to the early stages of this work. This work was supported by grants from the BBSRC and the Commission of the European Communities to S.J.F. and the Wellcome Trust to N.J.W. JWAA is grateful to the EPSRC for a studentship. This is a contribution from the Oxford Centre for Molecular Sciences, which is supported by the BBSRC, EPSRC and MRC.

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Authors and Affiliations

  1. Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom

    James W.A. Allen & Stuart J. Ferguson

  2. Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom

    Nicholas J. Watmough

  3. Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QT, United Kingdom

    Stuart J. Ferguson

Authors
  1. James W.A. Allen
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Correspondence to Nicholas J. Watmough or Stuart J. Ferguson.

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Allen, J., Watmough, N. & Ferguson, S. A switch in heme axial ligation prepares Paracoccus pantotrophus cytochrome cd1 for catalysis. Nat Struct Mol Biol 7, 885–888 (2000). https://doi.org/10.1038/82821

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